Application of a protein in the preparation of drugs for preventing and treating acute hepatopancreatic necrosis

Abstract

The present invention belongs to the field of disease prevention and control and aquaculture technology, relates to the characteristic therapeutic activity of known proteins, and specifically relates to the use of a protein in the preparation of a drug for preventing and treating acute hepatopancreatic necrosis. The amino acid sequence of the protein is shown in SEQ ID NO.1. The present invention has found that the protein has the ability to neutralize Vibrio Pir toxin, which can effectively block Pir toxin from penetrating the barrier to infect Japanese shrimp tissues, and enhance the resistance of Japanese shrimp to Vibrio Pir toxin, thereby achieving the prevention and treatment of acute hepatopancreatic necrosis.

Description

Technical Field

[0001] The invention belongs to the technical field of disease prevention and treatment and aquaculture, relates to the characteristic therapeutic activity of known proteins, and specifically relates to the application of a protein in the preparation of a drug for preventing and treating acute hepatopancreatic necrosis. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Acute hepatopancreatic necrosis (AHPND) has a very high mortality rate, usually causing the death of the entire shrimp population within a few days of onset. The disease mainly attacks the hepatopancreas of shrimp, causing severe organ damage and dysfunction, and ultimately causing a large number of deaths in Japanese marsupials. Related studies have shown that the main transmission route of AHPND is through infection with Vibrio carrying Pir toxin plasmids. The Pir toxins secreted by this bacterium are the main pathogenic factors of AHPND. Under natural conditions, the infection route of AHPND is that Vibrio enters the body of Japanese marsupials through the mouth and secretes Pir toxins to damage the tissues of Japanese marsupials. Although the chitin layer in the stomach of Japanese marsupials and the peritrophic membrane structure in the intestine, which is mainly composed of chitin and glycoproteins, are the first line of defense against foreign pathogens, the powerful invasive ability of Pir toxins can still effectively break through these defense barriers and invade the host. After penetrating the first barrier, Pir toxins spread rapidly in the host, interfering with normal physiological functions and causing extremely high mortality.

[0004] Although the pathogenic mechanism of AHPND has not been fully elucidated, there are some means to prevent it. For example, by using antibiotics or disinfectants, this method has limited effect and cannot be widely used because it will lead to the emergence of drug-resistant strains; by improving environmental factors such as temperature, pH and salinity, the water quality environment is adjusted to conditions that are not conducive to the growth of pathogens; by using probiotics to regulate the intestinal microbiome of Japanese shrimp, enhance its overall immune capacity and survival rate, etc. However, these methods are not targeted at the disease of AHPND or the pathogenic factor Pir toxin, so it is crucial to find a prevention and treatment method for AHPND. Summary of the invention

[0005] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide a protein for the preparation of a drug for the prevention and treatment of acute hepatopancreatic necrosis. The present invention has found that a protein originally derived from Japanese shrimp ( Marsupenaeus japonicus) has the ability to neutralize Vibrio Pir toxin, which can effectively block Pir toxin from penetrating the barrier and infecting the tissues of Japanese shrimp, and enhance the resistance of Japanese shrimp to Vibrio Pir toxin, thereby achieving the prevention and treatment of acute hepatopancreatic necrosis.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] In a first aspect, a protein is used in the preparation of a drug for preventing and treating acute hepatopancreatic necrosis, wherein the drug comprises the protein or a derivative thereof, and the amino acid sequence of the protein is shown in SEQ ID NO.1.

[0008] The protein of the present invention can be synthesized artificially, or its coding gene can be synthesized first and then expressed biologically, or it can be sent to a biological company for synthesis or the entire sequence can be synthesized using an automatic peptide synthesizer, or it can be obtained by preparing and using polyclonal or monoclonal antibodies of the protein and isolating and purifying it in Japanese shrimp.

[0009] The protein of the present invention may be in the form of a natural protein, a recombinant protein or a synthetic polypeptide, or may be chemically or physically modified on the basis of a natural or recombinant protein to improve its stability, activity or targeting in the intestine, which is not specifically limited here.

[0010] The protein derivative of the present invention is a product that can produce the protein. In some embodiments, the protein derivative can be an engineered bacterium, a recombinant expression vector or a nucleic acid molecule encoding the protein. The recombinant expression vector contains the nucleic acid molecule, and the engineered bacterium contains the recombinant expression vector. Specifically, the engineered bacterium can be a eukaryotic bacterium or a prokaryotic bacterium, such as Agrobacterium, yeast, Escherichia coli, etc. Specifically, the recombinant expression vector is obtained by effectively connecting the nucleic acid molecule to an expression vector, and the expression vector is a plasmid, a phage, a viral vector, a phagemid, a cosmid, a F cosmid, etc. Specifically, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA, etc. More specifically, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.2.

[0011] In some embodiments, the medicament comprises an effective amount of the protein. In some embodiments, the protein may be present in the medicament in a unit dosage form for administration.

[0012] In some embodiments, the drug is administered by direct feeding or feed addition. Specifically, the feed addition can be used as a feed additive, added to the feed, and fed to the aquatic animals together with the feed.

[0013] In some embodiments, the effective amount of the drug is: 10-20 μg of the protein is administered to each Japanese prawn.

[0014] In some embodiments, the drug is administered to an aquatic animal, and specifically, the aquatic animal is Japanese prawn.

[0015] In some embodiments, the drug is in the form of powder, soluble powder, granule premix, compound formulation, liquid coating, microcapsule embedding, etc. The specific form of the drug can be selected according to actual needs.

[0016] In some embodiments, the acute hepatopancreatic necrosis is caused by vibriosis caused by Vibrio parahaemolyticus.

[0017] In some embodiments, the causative agent of acute hepatopancreatic necrosis is Pir toxin. Specifically, the Pir toxin is PirAB toxin.

[0018] In some embodiments, the drug has one or more of the following effects:

[0019] (1) Neutralize Vibrio Pir toxin;

[0020] (2) Prevent Vibrio Pir toxin from invading the digestive tract tissues of aquatic animals;

[0021] (3) Improve the ability of aquatic animals to resist Vibrio containing Pir toxins;

[0022] (4) Improve the survival rate of aquatic animals infected with Vibrio Pir toxin.

[0023] The beneficial effects of the present invention are:

[0024] The protein provided by the present invention, whose amino acid sequence is shown in SEQ ID NO.1, binds to the chitin layer in the digestive tract of Japanese prawn and interacts with the Vibrio Pir toxin protein, thereby preventing the toxin protein from penetrating the chitin layer to infect the epithelial tissue and cause tissue damage. Specifically, the recombinant protein is expressed and purified by prokaryotic expression and orally fed to Japanese prawns, and the survival rate experiment, HE staining experiment, and immunofluorescence experiment, etc. verify that the recombinant protein has the ability to neutralize Vibrio Pir toxin and resist digestive tract infection.

[0025] The protein provided by the present invention, whose amino acid sequence is shown in SEQ ID NO.1, shows excellent effect in preventing and controlling AHPND. The protein can neutralize Pir toxin and reduce the amount of toxin infecting epithelial tissue and entering cells, and can be used for disease prevention and control in Japanese shrimp farming, and can effectively prevent and control AHPND. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0027] Figure 1 The protein (recombinant protein) obtained in Example 1 of the present invention;

[0028] Figure 2 This is a graph of the expression levels of the gene encoding the protein in Example 2 of the present invention in various tissues;

[0029] Figure 3 This is a diagram of the protein domain analysis in Example 2 of the present invention;

[0030] Figure 4 This is a distribution diagram of the protein in the stomach after being fed into the body of Japanese prawn in Example 2 of the present invention;

[0031] Figure 5 This is a diagram verifying the interaction between the protein and the Pir toxin in Example 3 of the present invention;

[0032] Figure 6 This is an analysis diagram of the extent to which Pir toxins invade into the tissues of Japanese shrimp after supplementation with the protein in Example 3 of the present invention;

[0033] Figure 7 This is a diagram showing tissue damage analysis of diseased Japanese marsupials after supplementation with the protein in Example 4 of the present invention;

[0034] Figure 8 It is the survival rate of the diseased Japanese shrimp after supplementing the protein in Example 4 of the present invention. DETAILED DESCRIPTION

[0035] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0036] The specific information of the amino acid sequence of the protein described in the following examples is as follows:

[0037] (a) Sequence characteristics

[0038] Length: 276 amino acids;

[0039] Type: amino acid;

[0040] Chain type: single chain;

[0041] (b) Molecule type: protein

[0042] (c) Original source: Japanese shrimp ( Marsupenaeus japonicus )

[0043] (d) Sequence description: Details are as follows:

[0044] MRSFNVLLLVAIGLAVVTAEEGSRGERGVTPDNFPYSHLCEDRPDKFICANCKTLVMCVKGQAFTRRCIENHFCAMKSEFGGSVCYPGEPAACTCQTANSFQVDPYDPQRFFACKNVGSKPEGYKCPDGMTFDQGTAQCK GGSSDASQCTMSGTFANPTNCSEYNKCIPLSSGWLQKSFTCNSGLMFNEKKGACEDPCHYQFVCQQEGRYPDLLNRRNYFECYVMGGKMTQDRFQCPDGYKWKVESAGVGNCVEDHDEDDGDYPFSRCYIPDGFCP, such as SEQ Shown as ID NO.1.

[0045] The specific information of the nucleic acid molecule sequence encoding the protein described in the following examples is as follows:

[0046] (a) Sequence characteristics

[0047] Length: 831 bases;

[0048] Type: Nucleic acid;

[0049] Chain type: single chain;

[0050] (b) Molecule type: nucleic acid;

[0051] (c) Original source: Japanese shrimp ( Marsupenaeus japonicus );

[0052] (d) Sequence description: Details are as follows:

[0053] atgaggtctttcaacgttcttcttctggtagccattggacttgccgttgtcacggcagaagagggctcgcgaggtgaacgcggcgttactcccgataacttcccgtactcgcatctatgtgaggacaggcctgataaattcatttgtgcaaactgcaagactctggtcatgtgtgtgaagggtcaagctttcactcgtcgctgcatcgagaatcatttctgtgcaatgaagagcgagtttggaggcagtgtatgctacccgggagaacctgcggcatgcacctgtcagactgccaattccttccaagtagacccttatgatccacagaggttcttcgcttgcaagaacgttggatctaaacccgagggctacaagtgccccgatggaatgactttcgaccaaggcaccgcacagtgcaagggtggaagtagcgatgcgtcacaatgcacgatgtcgggtaccttcgctaatcctactaattgtagtgaatacaacaagtgcattcctctgagcagtggatggctgcagaagtcgttcacgtgcaacagtggcttgatgttcaacgagaaaaagggggcgtgtgaggacccttgccactaccagttcgtgtgccaacaggagggacgctacccagacttgctgaacaggcgtaattacttcgaatgctacgtaatgggtggcaagatgacccaggaccgattccagtgccctgacggctacaagtggaaggttgaatccgcaggagtggggaattgtgtggaggaccatgacgaggacgacggtgattaccccttcagtcgatgctacatccctgatggcttttgtccctag, as shown in SEQ ID NO.2.

[0054] Example 1 Amplification and expression of the protein mature peptide sequence.

[0055] RNA from the intestinal tissue of Japanese shrimp was extracted using TRIzol Reagent, and then reverse transcribed into cDNA (as a template) using a reverse transcription kit (Novozyme, Nanjing, China, R323-01). Expression primers were designed according to SEQ ID NO. 2 provided by the present invention:

[0056] F1: 5'-GATCTGGTTCCGCGTGGATCCATGAGGTCTTTTCAACGTTTCTTCTTC-3' (SEQ IDNO.3);

[0057] R1: 5'-GATCTGGTTCCGCGTGGATCCCTAGGGACAAAAGCCATCAGGG-3' (SEQ ID NO.4), containing BamH I and Xho I restriction sites.

[0058] The sequence encoding the mature peptide of the protein was amplified by the above primers and recovered by gel. Subsequently, the pGEX-4T-1 plasmid was double-digested with BamH I and Xho I, and the target sequence was connected to the plasmid by homologous recombination. The recombinant vector was transformed into the Escherichia coli Rosetta (DE3) strain, and 0.2 mM isopropyl-β-D-thiogalactoside (IPTG) was used to induce expression at 28°C for 6 hours. Since the protein is an insoluble protein, it needs to be dissolved in 8 M urea at 28°C and 160 rpm overnight, and the supernatant was taken after centrifugation at 12000 rpm and lysed in PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mMKH 2 PO 4 , pH 7.4) and then dialyzed to obtain the target protein, i.e., the recombinant protein. Figure 1 The glutathione-S-transferase protein with a GST tag expressed in the pGEX-4T-1 plasmid was used as the control protein in Examples 1, 2, 3 and 4.

[0059] Example 2 Tissue distribution of mRNA levels and protein domain analysis and distribution in the digestive tract

[0060] After RNA was extracted from various tissues of Japanese shrimp and converted into cDNA, real-time fluorescence quantitative PCR (qPCR) was used to detect the expression level of the gene encoding the protein in various tissues. The primer sequences are:

[0061] F2: 5'-CAGACTTGCTGAACAGGCGTAA-3' (SEQ ID NO.5)

[0062] R2: 5'-TCCTCGTCATGGTCCTCCAC-3' (SEQ ID NO.6)

[0063] β-actin was used as an internal reference, and the primer sequences were:

[0064] F3: 5'-AGTAGCCGCCCTGGTTGTAGAC-3' (SEQ ID NO.7)

[0065] R3: 5'-TTCTCCATGTCGTCCAGT-3' (SEQ ID NO.8)

[0066] The qPCR program was set as follows: 94°C for 5 minutes, 94°C for 10 seconds, 60°C for 1 minute, and 40 cycles. The melting curve was measured from 65°C to 95°C at 1°C intervals.

[0067] The test results showed that the gene encoding the protein was expressed most highly in gastric tissue, indicating that the protein may play a role in the stomach, e.g. Figure 2 shown.

[0068] The amino acid sequence of the protein (SEQID NO.1) was predicted by the online SMART tool (https: / / smart.embl.de / ), and it was found that it contains three chitin-binding domains. Since the chitin layer in the stomach of Japanese shrimp and the peritrophic membrane structure in the intestine, which is mainly composed of chitin and glycoproteins, contain a large amount of chitin, the protein may have the ability to bind to the chitin layer, such as Figure 3 shown.

[0069] Then, 20 μg of recombinant protein was orally fed to Japanese prawns, and the localization of the recombinant protein in the stomach was detected 6 hours after feeding. After the experimental animal treatment, the stomach tissues of two groups of Japanese prawns were fixed in Davidson's fixative (30% ethanol, 22% formalin and 11.5% acetic acid) for 24 hours, and then immersed in 15% and 30% sucrose solutions for 24 hours for dehydration. After that, they were placed in the embedding medium and immersed overnight at 4 ℃ and then transferred to -20 ℃ for half an hour. The wax blocks of the embedded tissues were cut into wax slices with a thickness of 10 μm using a slicer (Leica CM 1860), and the wax slices were attached to the slides and dried at 60 ℃ for half an hour. Then, the protein was labeled with Gst primary antibody (mouse) and Dylight 649-labeled fluorescent secondary antibody (goat anti-mouse, 1:500 dilution), chitin was labeled with FITC-WGA, and cell nuclei were labeled with DAPI. The localization of the protein and chitin in the stomach was observed under a confocal microscope (Leica STELLARIS 5, Germany). The results showed that the protein can bind to the chitin layer in the intestine, such as Figure 4 shown.

[0070] Example 3 Verification of the interaction between the protein and Pir toxin and the change in the amount of toxin penetrating the chitin layer after feeding the protein

[0071] Yeast two-hybrid experiments were performed using the Matchmaker Gold yeast two-hybrid system (630495, Clontech, California, USA). The fragment encoding the toxin protein PirA and the fragment encoding the protein were ligated to the pGADT7 and pGBKT7 plasmids, respectively. A pair of plasmids were transformed into Y2H yeast cells and cultured and screened on DDO (SD / -Leu / -Trp) medium. The screened positive clone strains were inoculated on QDO / X (SD / -Ade / -Leu / -Trp / -His / Xa-gal) medium for further screening. Strains that can grow on DDO medium and grow as blue plaques on QDO / X medium indicate that there is an interaction. The results show that there is an interaction between the protein and the Pir toxin, such as Figure 5 shown.

[0072] In this example, the Japanese shrimp lived in a room temperature non-circulating water environment (the oxygenation device was provided by SUNSUN, China, and the seawater was provided by the Faguo Agricultural Products Store in Jimo District). The stocking density was set at 40 Japanese shrimp / m 3 In this environment, various water indicators (pH, ammonia nitrogen concentration, dissolved oxygen, etc.) will change over time to simulate the environmental changes that may occur during the breeding process. After oral feeding of the recombinant protein (10 μg / Penaeus japonicus) for 24 hours, the Pir toxin was orally fed into the body of Penaeus japonicus, and the Pir toxin was obtained by Example 2. Figure 4 The co-localization of Pir toxin and recombinant protein on the chitin layer and the changes in the amount of Pir toxin that penetrated the chitin layer and entered the underlying tissue were observed by the method of . The results showed that feeding the recombinant protein in advance could reduce the amount of toxin that penetrated the chitin layer and entered the tissue, indicating that the recombinant protein has the effect of resisting Pir toxin from infecting tissues. Figure 6 shown.

[0073] Example 4 Analysis of tissue damage and survival rate of infected Japanese shrimp after feeding the protein

[0074] After 24 h of oral feeding of the recombinant protein (10 μg / Penaeus japonicus), the bacterial suspension of Vibrio parahaemolyticus was added to the culture tank of Penaeus japonicus in proportion. After 48 h of treatment, the hepatopancreas tissue of Penaeus japonicus was collected and fixed with Davidson's AFA fixative (30% ethanol, 22% formalin and 11.5% acetic acid). After 24 h of fixation, the tissue was dehydrated, embedded in paraffin, sliced ​​at a thickness of 7 μm, and stained with hematoxylin and eosin (H&E). It was observed under a BX51 microscope (Olympus, Tokyo, Japan) and photographed using a DP70 digital camera system (Olympus). The results showed that after the pathogenic Vibrio infected Penaeus japonicus, the hepatopancreas tissue showed enlargement, hepatic duct atrophy, cell necrosis and a large number of blood cell infiltration. It can be found that feeding the recombinant protein in advance can reduce the damage of the hepatopancreas tissue of Penaeus japonicus after pathogenic bacteria infection, such as Figure 7 shown.

[0075] In order to reveal the role of the recombinant protein in resisting pathogenic Vibrio infection, a survival experiment was conducted to record the number of surviving Japanese shrimp for 6 consecutive days. The results showed that feeding the recombinant protein in advance can increase the survival rate of Japanese shrimp when infected with pathogenic bacteria, indicating that the protein has the effect of resisting pathogenic bacteria infection, such as Figure 8 shown.

[0076] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of a protein in the preparation of a drug for preventing and treating acute hepatopancreatic necrosis, the drug comprising the protein, the amino acid sequence of the protein being as shown in SEQ ID NO.1; The drug is administered to the Japanese shrimp.

2. The use according to claim 1, characterized in that: The medicine also includes a derivative product of the protein, which is an engineered bacterium, a recombinant expression vector or a nucleic acid molecule encoding the protein, the recombinant expression vector contains the nucleic acid molecule, and the engineered bacterium contains the recombinant expression vector.

3. The use according to claim 2, characterized in that: The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

2.

4. The use according to claim 1, characterized in that: The drug is administered by direct feeding or adding to feed.

5. The use according to claim 1, characterized in that: The effective amount of the drug is: 10-20 μg of the protein is administered to each Japanese prawn.

6. The use according to claim 1, characterized in that: The medicine is in the form of soluble powder, granule premix, compound preparation, liquid coating or microcapsule embedding.

7. The use according to claim 1, characterized in that: The acute hepatopancreatic necrosis is caused by Vibrio parahaemolyticus; Or, the causative factor of the acute hepatopancreatic necrosis is Pir toxin.

8. The use according to claim 1, characterized in that: The drug has one or more of the following effects: (1) Neutralize Vibrio Pir toxin; (2) Prevent Vibrio Pir toxin from invading the digestive tract tissues of aquatic animals; (3) Improve the ability of aquatic animals to resist Vibrio containing Pir toxins; (4) Improve the survival rate of aquatic animals infected with Vibrio Pir toxin.

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